The Rev-RRE interaction and the subsequent transport of intron-containing viral mRNA mediated by the cellular export receptor CRM1 are essential to the replication of the HIV-1 retrovirus. The current model for oligomeric Rev binding to its cognate RRE is inadequate to fully explain the function of the regulatory protein Rev. While the importance of Rev oligomerization has been previously established, there is no clear molecular basis for understanding the requirements for nuclear transport and the interactions with the nuclear export factor CRM1. We propose to address several mechanistic and structural questions aiming to bridge the gap between Rev-oligomerization, RRE-binding and CRM1-mediated RNA export. Our integrated biochemical, biophysical, and NMR structural studies are centered on the molecular mechanisms by which the regulatory HIV-1 protein Rev attains its biologically active structures. A comprehensive model of how Rev works could potentially facilitate rational drug design targeting this essential viral gene. Our central hypothesis is that Rev function requires: 1) a defined structure and 2) oligomerization ability to serve as a substrate for the cellular export machinery. Monomeric Rev is intrinsically disordered. An on-pathway, partially folded Rev intermediate and the ordered oligomerization nucleus interconvert;a well-ordered Rev structure requires stabilization by either oligomeric binding to RRE, binding to other cofactors, or fibrillation. The proposed development of a comprehensive structure-activity relationship will also test the hypothesis whether modulation of Rev activity could confer positive selection advantages under either host immune responses or antiretroviral therapy that in turn can be linked to the slow progression of AIDS and escape from the immune system. The fundamental question concerning the export activity of the HIV-1 regulatory protein Rev becomes: What are the molecular and mechanistic features connecting Rev structure, Rev oligomerization, oligomeric Rev-RRE assembly and the interaction with cellular CRM1 and Ran-GTP? We propose to answer this question by pursuing the following Specific Aims: 1) Understand the function and molecular mechanism of Rev oligomerization. 2) Elucidate the structures and dynamics of monomeric and oligomeric Rev and of its stem II RRE substrate. 3) Develop mechanistic and structural models for CRM1/Ran-GTP-mediated nuclear export involving oligomeric Rev-RRE substrate complexes. PUBLIC HEALTH RELEVANCE: The HIV retrovirus encodes a small number of proteins and thus must exploit specific cellular cofactors for successful replication. We will investigate the interactions between the essential HIV regulatory protein Rev and cellular proteins involved in nuclear export of viral RNA. Understanding the molecular details of Rev-dependent RNA transport will provide new avenues to therapies that aim at viral eradication.